Summary: Francisella tularensis (FT), the causative agent for tularemia, can infect humans by a number of routes, including vector-borne transmission. However, it is inhalation of the bacterium, and the resulting pneumonic tularemia, that represents the most dangerous form of disease. This is due to the short incubation time (3-5 days), non-specific symptoms, and a high mortality rate (greater than 80%) in untreated individuals. Furthermore, FT has been weaponized by both the United States and the former Soviet Union making it a viable candidate for use as a biological weapon. Despite over 80 years of research on FT around the world, very little is understood about the dynamic interaction of this bacterium with the host, especially following aerosol infection. My laboratory has established that, similarly to murine cells, human dendritic cells and macrophages are acutely susceptible to infection with FT, but fail to produce pro-inflammatory cytokines or undergo maturation. Further, virulent FT actively interferes with the ability of human DC and macrophages to respond to secondary stimuli. Understanding the mechanism by which FT actively suppresses DC and macrophage function is a central directive of my laboratory. We are tackling this directive in two different ways. Specific Aim 1: We are analyzing the role Francisella lipids play in mediating anti-inflammatory responses. Structures present on the surface of bacteria are the first components encountered by the host cell. Thus, it is possible that, in the context of FT infections, these structures contribute to the early, rapid suppression of human dendritic cells. Bacterial lipids represent one such structure. We have recently identified one of the active lipid species present in FT that inhibit inflammation. We have tested a synthetic version of this lipid and confirmed that it also impairs inflammatory responses. We have identified one of the receptors utilized by both the intact bacterium and purified lipid as well as a host signaling molecule required to promote immunosuppressive responses. Importantly, we have also demonstrated that FT lipids can limit pathogenic inflammatory responses driven by other infectious agents in vitro, including Dengue virus. We have screened FT lipids (including synthetic liposomes) for off target effects in vivo following delivery via multiple routes and are preparing to examine their efficacy against pathogen mediated inflammation in vivo. We have filed a patent application for use of FT lipids as novel anti-inflammatory therapeutics. We are currently identifying the other lipids present in FT that contribute to suppression of inflammation and additional mechanisms by which they interfere with functions in human cell. We have generated a lipid mutant and identified a requirement for a specific species of FT lipid in evasion, but not suppression, of pro-inflammatory responses in human cells. Specific Aim 2: We are exploring the role of carbohydrates associated with the outer surface of FT in directing immunosuppressive programs in human cells. The major outer surface carbohydrate structure of FT is the O-Antigen (O-Ag) capsule. Typically, capsules are thought to simply cover up proteins present on the bacterial surface that could stimulate an inflammatory response. However, our data demonstrates that FT capsule directly inhibits pro-inflammatory responses in human cells. Further, we have demonstrated that capsule influences specific metabolic pathways in the host the modulate cell health and inflammatory responses. Utilizing mutants with specific defects in capsule synthesis along with purified FT capsule, we are currently identifying the specific receptors and host signaling pathways modulated by capsule to initiate an anti-inflammatory program in human cells.